scholarly journals CRUNC: a cryopreservation method for unencapsulated gemmae of Marchantia polymorpha

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10174
Author(s):  
Hitomi Takahashi ◽  
Yutaka Kodama
Keyword(s):  
Liquid Nitrogen ◽  
Marchantia Polymorpha ◽  
Wild Type ◽  
High Recovery ◽  
Simple Method ◽  
High Recovery Rate ◽  
Genetic Modifications ◽  
Transgenic Lines ◽  
Cryopreservation Method

Genetic modifications such as mutation and transformation are powerful tools to study the function of genes and proteins in the model liverwort Marchantia polymorpha, but maintaining the resulting germplasm requires a practical, reliable method. Cryopreservation methods allow researchers to maintain mutant and transgenic lines of M. polymorpha. To date, two methods have been developed for cryopreservation of M. polymorpha gemmae: in the first method, unencapsulated gemmae are stored in liquid nitrogen at −­196 °C, and in the second method, encapsulated gemmae are stored in liquid nitrogen at −­196 °C or a deep freezer at −80 °C. In the present study, we developed a simple method named CRUNC (cr yopreservation of un en c apsulated gemmae), which can be used to store unencapsulated, dried gemmae of wild-type and transgenic M. polymorpha lines in liquid nitrogen and in freezers at −80 °C and −20 °C. Using the CRUNC method, we observed a high recovery rate (as high as 100%) and successful long-term (5 months) storage of the gemmae. Therefore, the CRUNC method is practical for maintaining valuable M. polymorpha germplasm.

scholarly journals Cryopreservation of Anopheles stephensi embryos

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Eric R. James ◽  
Yingda Wen ◽  
James Overby ◽  
Kristen Pluchino ◽  
Shane McTighe ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Life Cycle ◽  
Infectious Diseases ◽  
Anopheles Stephensi ◽  
Mass Storage ◽  
Genetic Studies ◽  
Transgenic Lines ◽  
Cryopreservation Method ◽  
Anopheline Mosquitoes

AbstractThe ability to cryopreserve mosquitoes would revolutionize work on these vectors of major human infectious diseases by conserving stocks, new isolates, lab-bred strains, and transgenic lines that currently require continuous life cycle maintenance. Efforts over several decades to develop a method for cryopreservation have, until now, been fruitless: we describe here a method for the cryopreservation of Anopheles stephensi embryos yielding hatch rates of ~ 25%, stable for > 5 years. Hatched larvae developed into fertile, fecund adults and blood-fed females, produced fully viable second generation eggs, that could be infected with Plasmodium falciparum at high intensities. The key components of the cryopreservation method are: embryos at 15–30 min post oviposition, two incubation steps in 100% deuterated methanol at − 7 °C and − 14.5 °C, and rapid cooling. Eggs are recovered by rapid warming with concomitant dilution of cryoprotectant. Eggs of genetically modified A. stephensi and of A. gambiae were also successfully cryopreserved. This enabling methodology will allow long-term conservation of mosquitoes as well as acceleration of genetic studies and facilitation of mass storage of anopheline mosquitoes for release programs.

scholarly journals Kriopreservasi untuk Konservasi Plasma Nutfah Tanaman: Peluang Pemanfaatannya di Indonesia

2016 ◽  
Vol 3 (2) ◽  
pp. 80
Author(s):  
Semuel Leunufna
Keyword(s):  
Ex Situ Conservation ◽  
Germplasm Collection ◽  
Ex Situ ◽  
Seed Plants ◽  
High Recovery ◽  
High Recovery Rate ◽  
Long Term Preservation ◽  
Collection Methods

<p>Increasing rate of plant germplasm lost in<br />Indonesia has promoted the implementation of various<br />methods for their conservation. Cryopreservation is a technique<br />applicable for a long-term preservation (base collection)<br />of plants possessing non-orthodox (recalcitrant and<br />semi-recalcitrant) seeds and those propagated vegetatively.<br />The technique can be used as an alternative method for<br />orthodox seed plants preservation in the ex situ conservation<br />system. Although field and in vitro collection methods can<br />be applied for the non-orthodox seed plants, a number of<br />disadvantages possesed by these methods, especially in the<br />tropics or the developing countries, deny their use for the<br />establishment of a long-term germplasm collection. Successful<br />implementation of the cryopreservation technique is<br />supported by the development of protocols, which are able<br />to provide a high recovery rate for species understudy, using<br />vitrification based methods which are simple, economical,<br />applicable to complex organs, and able to implement a high<br />number of explants per experiment. The availability of infrastructures<br />including in vitro culture laboratories, continue<br />supply of liquid nitrogen is highly supporting the use of<br />cryopreservation technique in Indonesia.</p>

scholarly journals Efficient Cryopreservation of Populus tremula by In Vitro-Grown Axillary Buds and Genetic Stability of Recovered Plants

Plants ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 77
Author(s):  
Elena O. Vidyagina ◽  
Nikolay N. Kharchenko ◽  
Konstantin A. Shestibratov
Keyword(s):  
Survival Rate ◽  
Axillary Buds ◽  
Long Term Storage ◽  
Average Survival ◽  
Transgenic Lines ◽  
Ssr Loci ◽  
One Step ◽  
The One

Axillary buds of in vitro microshoots were successfully frozen at –196 °C by the one-step freezing method using the protective vitrification solution 2 (PVS2). Microshoots were taken from 11 transgenic lines and three wild type lines. Influence of different explant pretreatments were analyzed from the point of their influence towards recovery after cryopreservation. It was found out that the use of axillary buds as explants after removal of the apical one increases recovery on average by 8%. The cultivation on growth medium of higher density insignificantly raises the regenerants survival rate. Pretreatment of the osmotic fluid (OF) shows the greatest influence on the survival rate. It leads to the increase in survival rate by 20%. The cryopreservation technology providing regenerants average survival rate of 83% was developed. It was based on the experimental results obtained with explant pretreatment. Incubation time in liquid nitrogen did not affect the explants survival rate after thawing. After six months cryostorage of samples their genetic variability was analyzed. Six variable simple sequence repeat (SSR) loci were used to analyze genotype variability after the freezing-thawing procedure. The microsatellite analysis showed the genetic status identity of plants after cryopreservation and of the original genotypes. The presence of the recombinant gene in the transgenic lines after cryostorage were confirmed so as the interclonal variation in the growth rate under greenhouse conditions. The developed technique is recommended for long-term storage of various breeding and genetically modified lines of aspen plants, as it provides a high percentage of explants survival with no changes in genotype.

An ER–Golgi Tethering Factor SLOH4/MIP3 Is Involved in Long-term Heat Tolerance of Arabidopsis

2020 ◽  
Author(s):  
Kazuho Isono ◽  
Ryo Tsukimoto ◽  
Satoshi Iuchi ◽  
Akihisa Shinozawa ◽  
Izumi Yotsui ◽  
...  
Keyword(s):  
Heat Stress ◽  
Heat Tolerance ◽  
Secretory Proteins ◽  
Wild Type ◽  
Additional Function ◽  
Transcript Levels ◽  
Unfolded Protein ◽  
In The Wild ◽  
Protein Response

Abstract Plants are often exposed not only to short-term (S-) heat stress but also to diurnal long-term (L-) heat stress over several consecutive days. To reveal the mechanisms underlying L-heat stress tolerance, we here used a forward genetic screening for sensitive to long-term heat (sloh) mutants and isolated sloh4. The mutant was hypersensitive to L- but not S-heat stress. The causal gene of sloh4 was identical to MIP3 encoding a member of the MAIGO2 (MAG2) tethering complex, which is composed of the MAG2, MIP1, MIP2, and MIP3 subunits and is localized at the endoplasmic reticulum (ER) membrane. Although sloh4/mip3 was hypersensitive to L-heat stress, the sensitivity of the mag2-3 and mip1–1 mutants was similar to that of the wild type. Under L-heat stress, the ER stress and the following unfolded protein response (UPR) were more pronounced in sloh4 than in the wild type. Transcript levels of bZIP60-regulated UPR genes were strongly increased in sloh4 under L-heat stress. Two processes known to be mediated by INOSITOL REQUIRING ENZYME1 (IRE1)—accumulation of the spliced bZIP60 transcript and a decrease in the transcript levels of PR4 and PRX34, encoding secretory proteins—were observed in sloh4 in response to L-heat stress. These findings suggest that misfolded proteins generated in sloh4 under L-heat stress may be recognized by IRE1 but not bZIP28, resulting in initiation of the UPR via activated bZIP60. Therefore, it would be possible that only MIP3 in MAG2 complex has an additional function in L-heat tolerance, which is not related to the ER–Golgi vesicle tethering.

scholarly journals Effect of Spaceflight on Tomato Seed Quality and Biochemical Characteristics of Mature Plants

Horticulturae ◽  
2021 ◽  
Vol 7 (5) ◽  
pp. 89
Author(s):  
Elena Dzhos ◽  
Nadezhda Golubkina ◽  
Marina Antoshkina ◽  
Irina Kondratyeva ◽  
Andrew Koshevarov ◽  
...  
Keyword(s):  
Seed Quality ◽  
Space Station ◽  
Field Conditions ◽  
Tomato Seed ◽  
Tomato Plants ◽  
Biochemical Characteristics ◽  
Tomato Seeds ◽  
Genetic Modifications ◽  
Total Antioxidant

Intensive space exploration includes profound investigations on the effect of weightlessness and cosmic radiation on plant growth and development. Tomato seeds are often used in such experiments though up to date the results have given rather vague information about biochemical changes in mature plants grown from seeds subjected to spaceflight. The effect of half a year of storage in the International Space Station (ISS) on tomato seeds (cultivar Podmoskovny ranny) was studied by analyzing the biochemical characteristics and mineral content of mature plants grown from these seeds both in greenhouse and field conditions. A significant increase was recorded in ascorbic acid, polyphenol and carotenoid contents, and total antioxidant activity (AOA), with higher changes in the field conditions compared to greenhouse. Contrary to control plants, the ones derived from space-stored seeds demonstrated a significant decrease in root AOA. The latter plants also showed a higher yield, but lower content of fruit dry matter, sugars, total dissolved solids and organic acids. The fruits of plants derived from space-stored seeds demonstrated decreased levels of Fe, Cu and taste index. The described results reflect the existence of oxidative stress in mature tomato plants as a long-term consequence of the effect of spaceflight on seed quality, whereas the higher yield may be attributed to genetic modifications.

scholarly journals Overexpression of vesicle-associated membrane protein PttVAP27-17 as a tool to improve biomass production and the overall saccharification yields in Populus trees

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Madhavi Latha Gandla ◽  
Niklas Mähler ◽  
Sacha Escamez ◽  
Tomas Skotare ◽  
Ogonna Obudulu ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Biomass Production ◽  
Enzymatic Saccharification ◽  
Dry Weight ◽  
Populus Tremula ◽  
Transgenic Trees ◽  
Wild Type ◽  
Glucose Yield ◽  
Transgenic Lines ◽  
Wild Type Control

Abstract Background Bioconversion of wood into bioproducts and biofuels is hindered by the recalcitrance of woody raw material to bioprocesses such as enzymatic saccharification. Targeted modification of the chemical composition of the feedstock can improve saccharification but this gain is often abrogated by concomitant reduction in tree growth. Results In this study, we report on transgenic hybrid aspen (Populus tremula × tremuloides) lines that showed potential to increase biomass production both in the greenhouse and after 5 years of growth in the field. The transgenic lines carried an overexpression construct for Populus tremula × tremuloides vesicle-associated membrane protein (VAMP)-associated protein PttVAP27-17 that was selected from a gene-mining program for novel regulators of wood formation. Analytical-scale enzymatic saccharification without any pretreatment revealed for all greenhouse-grown transgenic lines, compared to the wild type, a 20–44% increase in the glucose yield per dry weight after enzymatic saccharification, even though it was statistically significant only for one line. The glucose yield after enzymatic saccharification with a prior hydrothermal pretreatment step with sulfuric acid was not increased in the greenhouse-grown transgenic trees on a dry-weight basis, but increased by 26–50% when calculated on a whole biomass basis in comparison to the wild-type control. Tendencies to increased glucose yields by up to 24% were present on a whole tree biomass basis after acidic pretreatment and enzymatic saccharification also in the transgenic trees grown for 5 years on the field when compared to the wild-type control. Conclusions The results demonstrate the usefulness of gene-mining programs to identify novel genes with the potential to improve biofuel production in tree biotechnology programs. Furthermore, multi-omic analyses, including transcriptomic, proteomic and metabolomic analyses, performed here provide a toolbox for future studies on the function of VAP27 proteins in plants.

Evaluation of long-term frozen storage of plasma for measurement of high-density lipoprotein and its subfractions by precipitation

1990 ◽  
Vol 36 (5) ◽  
pp. 783-788 ◽  
Author(s):  
M N Nanjee ◽  
N E Miller
Keyword(s):  
High Density Lipoprotein ◽  
Liquid Nitrogen ◽  
Frozen Storage ◽  
Density Lipoprotein ◽  
High Density ◽  
Long Term Stability ◽  
Long Term Storage ◽  
Different Temperatures ◽  
Edta Plasma

Abstract The concentration of high-density lipoprotein cholesterol (HDL-C) in plasma is now established as an independent risk factor for coronary heart disease, but more data are needed on the relative risk-predictive powers of different HDL subclasses. For epidemiologic and clinical purposes, isolation of HDL from other lipoproteins and separation of its two major subclasses, HDL2 and HDL3, are performed most conveniently by precipitation. Although storage of plasma is commonly necessary, little information is available on the long-term stability of HDL subclasses at different temperatures. Therefore, we quantified HDL-C, HDL2-C, and HDL3-C by dual precipitation with heparin-MnCl2/15-kDa dextran sulfate (H-M/DS) in samples of EDTA-plasma from 93 healthy subjects, after storage for one to 433 days at -20 degrees C, at -70 degrees C, or in liquid nitrogen (-196 degrees C). Fourteen samples (15%) were stored for a year or longer. At -20 degrees C, HDL-C decreased by 4.8% per year and HDL3-C decreased by 6.9% per year (P = 0.002 for both variables) relative to results obtained with samples stored in liquid nitrogen; total cholesterol, HDL2-C, and triglyceride did not change significantly at this temperature. When stored at -70 degrees C, none of the lipids showed any change relative to results obtained with liquid nitrogen. Thus, long-term storage of EDTA-plasma at -20 degrees C is unsuitable for subsequent quantification of HDL-C and its subclasses by H-M/DS dual precipitation. Storage at -70 degrees C is preferable, and is as reliable as storage in liquid nitrogen.

scholarly journals A Replication-Defective Gammaherpesvirus Efficiently Establishes Long-Term Latency in Macrophages but Not in B Cells In Vivo

2008 ◽  
Vol 82 (17) ◽  
pp. 8500-8508 ◽  
Author(s):  
Haiyan Li ◽  
Kazufumi Ikuta ◽  
John W. Sixbey ◽  
Scott A. Tibbetts
Keyword(s):  
B Cells ◽  
Viral Genome ◽  
Lytic Replication ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virus Latency

ABSTRACT Murine gammaherpesvirus 68 (γHV68 or MHV68) is genetically related to the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), providing a useful system for in vivo studies of the virus-host relationship. To begin to address fundamental questions about the mechanisms of the establishment of gammaherpesvirus latency, we previously generated a replication-defective γHV68 lacking the expression of the single-stranded DNA binding protein encoded by orf6. In work presented here, we demonstrate that this mutant virus established a long-term infection in vivo that was molecularly identical to wild-type virus latency. Thus, despite the absence of an acute phase of lytic replication, the mutant virus established a chronic infection in which the viral genome (i) was maintained as an episome and (ii) expressed latency-associated, but not lytic replication-associated, genes. Macrophages purified from mice infected with the replication-defective virus harbored viral genome at a frequency that was nearly identical to that of wild-type γHV68; however, the frequency of B cells harboring viral genome was greatly reduced in the absence of lytic replication. Thus, this replication-defective gammaherpesvirus efficiently established in vivo infection in macrophages that was molecularly indistinguishable from wild-type virus latency. These data point to a critical role for lytic replication or reactivation in the establishment or maintenance of latent infection in B cells.

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